Gene-silencing by siRNAs is a powerful technology for manipulating gene expression and a potential therapeutic strategy for treating human diseases. Canonical siRNAs are ˜21-nucleotide (nt) small RNAs that mimic products of Dicer processed double strand RNAs and can be incorporated into the RNA-induced-silencing-complex (RISC) to trigger the degradation of mRNA targets that contain highly complementary sequences (Elbashir 2001). Canonical siRNAs are designed to resemble the biogenesis intermediates of microRNAs (miRNA), a family of endogenous small RNAs that can repress the translation of target mRNAs that contain fully or partially complementary sequences. Therefore, siRNA and miRNA share the same functional machineries in the cell (Doench 2003; Zeng 2003).
The majority of miRNAs use Dicer to process the precursor-miRNAs (pre-miRNAs) to create 21 to 23-nt duplex RNAs that consist of one strand from the 5′ arm (5p) and one strand from the 3′ arm (3p). The 3′ end of each strand has an overhang of two nt. This duplex RNA is also referred to as miRNA/miRNA*(the dominant strand/the less abundant strand). Accordingly, siRNAs are designed as duplexes of antisense strand/sense strand (guide strand/passenger strand) RNAs that are 21-nt long, and have a 19 base pair dsRNA stem and an overhang of two nt at the 3′ end of each strand (siRNA, FIG. 1A). In contrast, similar duplexes that have overhangs of two nt at the 5′ end (hereafter referred to as reverse siRNA or rsiRNA, FIG. 1B) are thought to be mostly inactive (Elbashir 2001). DNA vector systems can also be used to express siRNAs as short hairpin RNAs (shRNAs, exp-shRNA), which can be used to express corresponding siRNAs in stable cell lines (McManus 2002; Brummelkamp 2002).
Several recent publications have revealed critical roles for loops, length of stems, and base pairing in the stem in exp-shRNA processing and silencing potency (Gu 2012; Herrera-Carrillo 2014; McIntyre 2011). In vitro T7 transcribed or chemically synthesized shRNAs (syn-shRNAs) were also shown to be potent RNAi triggers (Siolas 2005). The functional structure of syn-shRNAs was further characterized and the short stem version was named as short shRNAs (sshRNA), which are Dicer-independent (Ge 2012; Dallas 2012). Despite its extensive application as an effective gene manipulation reagent in research, the bright future of RNAi therapeutics is shadowed by growing evidence that many siRNAs have toxic side effects due to off-target activities of both the sense and anti-sense strands. These off-target effects will also produce biased research data (Jackson 2003). Therefore, siRNA molecules that have a potent on-target effect and lack off-target activities are highly desirable for both clinical and research applications. Despite extensive bodies of work accomplished in the past decade for this purpose, it remains a challenge to find an optimized siRNA for a specific target. Thus, there is a need for detailed parameters that can be used to effectively create optimal shRNAs that can be further processed into potent siRNAs.